变异催乳素受体Delta S2 PRLR与人类乳腺癌：Delta S2 PRLR对乳腺上皮细胞转录组及表观遗传学修饰的影响

A sizable body of evidence showed that prolactin (PRL) plays an important pathogenic role in reproductive cancers such as breast cancer, prostate cancer and ovarian cancer. PRL exerts its effects through binding to its receptor (PRLR) on the plasma membrane. Three isoforms of PRLR have been clarified so far in human. They are long form (LF), short form 1a (SF1a) and 1b (SF1b). We previously described a new variant of prolactin receptor (PRLR) in which the S2 subdomain of the extracelluar domain is missing (Delta S2). Moreover, each isoform of PRLR has its own Delta S2 variant. They are Delta S2 LF, Delta S2 SF1a and Delta S2 SF1b.Using bioluminescence resonance energy transfer (BRET) technology, we evidenced that the newly discovered Delta S2 variant has ability to be dimerized in the absence of PRL (constitutive dimerization) and initiated the cellular signal transduction as well. Furthermore, overexpression of Delta S2 LF in human breast epithelial cells induced cell proliferation. We hypothesized that Delta S2 PRLR services as an important regulator of growth of cells such as breast epithelial cells and of breast development, and that it plays an important pathogenic role in the initiation, progression and prognosis of human breast cancer. The aims of the study proposed here are: (1) to clarify the effect of Delta S2 LF, Delta S2 SF1a and Delta S2 SF1b on growth of human breast epithelial cells using adenovirus mediated gene transfer technology; (2) RNA sequencing (RNA-Seq) technology will be applied to detect the gene expression of breast epithelial cells in case of overexpression of Delta S2 PRLR in a genome wide manner (transcriptomics); (3) also, their effect on the epigenetic signature in human breast epithelial cells will be analyzed using chromatin immunoprecipitation assay (ChIP-Assay) and sequencing (ChIP-Seq) technology; (4) the relative expression level of each Delta S2 PRLR in human breast epithelial cells, as well as human breast tissue (normal and cancerous)will be examined using quantitative real time polymerase chain reaction (qRT-PCR) and western blot technology.

催乳素（PRL）与乳腺癌的形成、发展及预后关系密切。人催乳素受体(PRLR)主要分为催乳素长受体（LF）及两种短受体（SF1a及SF1b）。Delta S2 PRLR由申请人首次克隆并报道。长受体及两种短受体均有相应的Delta S2变体。实验证实，Delta S2 PRLR能够发生自发性二聚化作用,并启动相应的细胞信号转导。将Delta S2 LF cDNA转染给乳腺癌细胞，在没有PRL的情况下具有促进细胞增殖、激活β-casein基因启动子的作用。我们推测该变体在乳腺癌的发生过程中有重要意义。本研究将全面探讨各型Delta S 2PRLR对正常及癌变乳腺上皮细胞的增殖、转录组及表观遗传学修饰的影响，并检测各型Delta S2 PRLR与PRLR比值，重点比较正常乳腺上皮细胞与乳腺癌细胞之间、正常乳腺组织及乳腺癌组之间的差别。

催乳素（PRL）是调控乳腺上皮增殖分化的主要因素，PRL经过与靶细胞膜表面的催乳素受体（PRLR）而产生作用。大量研究证明PRL-PRLR信号途径参与了人类乳腺癌的形成。我们的研究发现，乳腺癌组织存在PRLR的变体，该变体其膜外区的一个亚区S2丢失（Delta S2 PRLR）。由于PRLR有一种长受体（LF）及两种短受体（SF1a及SF1b），三种受体主要区别仅在膜内区，其他部分完全一样。进一步研究表明，三种受体均存在其相应的Delta S2 变体，即Delta S2 LF、Delta S2 SF1a以及Delta S2 SF1b。本课题对这一新克隆的催乳素受体的变体的生物学特性、效应以及对人类乳腺癌细胞基因组的表观遗传学影响等进行了较全面的探讨。1、Delta S2 PRLR的生物学特性及生物学效应探讨。(1) Delta S2 PRLR 虽然膜外区S2亚区丢失，但蛋白合成后依然能够顺利到达细胞膜，符合膜受体的特征。(2) 变异受体依然能够发生二聚化，甚至产生自发性的二聚化。（3）相应的，具有促进乳腺癌细胞增殖的作用。2、乳腺癌组织催乳素受体水平的测定： Delta S2 LF在乳腺癌组织明显高于正常组织，Delta S2 SF1b水平则显著低于癌旁正常乳腺组织。3、 Delta S2 PRLR生物学作用。 （1）运用免疫共沉淀方法证实Delta S2与雌激素受体（ERα）、VDR以及ERK等有交互作用。（2）过表达Delta S2 PRLR能够引起人类乳腺上皮细胞包括乳腺癌细胞基因组表观遗传学的显著改变，例如引起p53基因组蛋白甲基化（H3K27Me3），解释了其引起乳腺癌细胞增殖的分子机制。（3）Delta S2 LF引起转录组即包括mRNA、snoRNA以及微小RNA等的改变，本次研究发现有27个肿瘤相关基因仅在过表达Delta S2 LF的细胞表达，发现部分凋亡相关基因，在Delta S2 LF过表达的细胞不再表达，此外，结果还表明Delta S2 LF能够引起某些miRNA的表达。（4）Delta S2 SF1a过表达能够引起乳腺癌细胞转录组的改变，基因本体学分析表明，在三大类别即生物过程、细胞组分、分子功能中，均有20项以上发生改变。